This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Raynaud Phenomenon (RP) is characterized by enhanced vasoconstriction in response to cold, emotional stress or exposure to vibrations. These responses are prevented by alpha2-adrenergic (alpha2-AR) antagonists. Several lines of evidence indicate that [unreadable]2C-AR subtype mediate the RP vasospastic attacks, but the cellular and molecular mechanisms underlying this effect remain unknown. In the preliminary investigation we found that alpha2C-AR accumulates in the endoplasmic reticulum at 37oC. Treatments interfering with the receptor export trafficking, but not with receptor internalization enhanced the plasma membrane alpha2C-AR levels. A similar enhancement was observed in cells exposed to low-temperature, indicating that cold increases the alpha2C-AR availability at the cell surface, supplementing the number of receptors interacting with local catecholamines and leading to exaggerated vasoconstriction. Further, we identified arginine based motifs embedded in the alpha2C-AR structure acting as endoplasmic reticulum retention motif at 30oC, but not at 37oC. The mechanisms mobilized by these retention motifs involve, at least in part, temperature-dependent interactions with [unreadable]-COP, a subunit of COPI vesicles regulating the retrograde traffic from Golgi to endoplasmic reticulum. Lastly, the alpha2C-AR plasma membrane levels were enhanced by treatment with HSP90 inhibitors at 37oC but not at 30oC. Further, the receptor interactions with the cytosolic HSP90 isoforms were temperature-dependent. Based on these preliminary data the central hypothesis of this application is that augmentation of the alpha2C-AR plasma membrane levels by low-temperature is due to specific RXR motifs embedded in its structure. These motifs are mediating receptor interactions with specific molecular chaperones in temperature-sensitive manner. Identification of these RXR motifs and the assisting chaperones will significantly advance the understanding of the pathological mechanisms underlying RP. To achieve these goals a combination of cell and molecular biology approaches will be used in HEK293T and vascular smooth muscle cells, aiming to elucidate the following problems: 1) identification of the retention motifs conferring temperature sensitivity to alpha2C-AR traffic;2) defining the molecular mechanisms mobilized by these retention motifs and 3) characterization of the HSP90 roles in the temperature-dependent regulation of alpha2C-AR transport to the cell surface. These specific aims are independent, but also interconnected and such organization warrants generation of new data on the cellular mechanisms leading to RP. It will also contribute to identification of cellular biomarkers for early detection of RP and it may provide foundation for designing more effective therapeutic strategies.